Most eukaryotic genes are discontinuous with proteins encoded by them, consisting of coding sequences (exons) interrupted by non-coding sequences (introns). After transcription into RNA, the introns are removed by splicing to generate the mature messenger RNA (mRNA). The splice points between exons are typically determined by consensus sequences that act as signals for the splicing process.
Structural features of introns and the underlying splicing mechanisms form the basis for classification of different kinds of introns. Since RNA splicing was first described, four major categories of introns have been recognized. Splicing of group I, group II, nuclear pre-mRNA, and tRNA introns can be differentiated mechanistically, with certain group I and group II introns able to be autocatalytically excised from a pre-RNA in vitro in the absence of any other protein or RNA factors. In the instance of the group I, group II and nuclear pre-mRNA introns, splicing proceeds by a two-step transesterification mechanism.
To illustrate, the nuclear rRNA genes of certain lower eukaryotes (e.g., Tetrahymena thermophila and Physarum polycephalum) contain group I introns. This type of intron also occurs in chloroplast, yeast, and fungal mitochondrial rRNA genes; in certain yeast and fungal mitochondrial mRNA; and in several chloroplast tRNA genes in higher plants. Group I introns are characterized by a linear array of conserved sequences and structural features, and are excised by two successive transesterifications. Splicing of the Tetrahymena pre-rRNA intron, a prototypic group I intron, proceeds by two transesterification reactions during which phosphate esters are exchanged without intermediary hydrolysis. Except for the initiation step, promoted by a free guanosine, all reactive groups involved in the transesterification reactions are contained within the intron sequences. The reaction is initiated by the binding of guanosine to an intron sequence. The unshared pair of electrons of the 3'-hydroxyl group of the bound guanosine can act as a nucleophile, attacking the phosphate group at the 5' exon-intron junction (splice site), resulting in cleavage of the precursor RNA. A free 3'-hydroxyl group is generated at the cleavage site (the end of the 5'exon) and release of the intron occurs in a second step by attack of the 5' exon's 3.sup.1 -hydroxyl group on the 3' splice site phosphate.
Group II introns, which are classed together on the basis of a conserved secondary structure, have been identified in certain organellar genes of lower eukaryotes and plants. The group II introns also undergo self-splicing reactions in vitro, but in this instance, a residue within the intron, rather than added guanosine, initiates the reaction. Another key difference between group II and group I introns is in the structure of the excised introns. Rather than the linear products formed during splicing of group I introns, spliced group II introns typically occur as lariats, structures in which the 5'-phosphoryl end of the intron RNA is linked through a phoshodiester bond to the 2'-hydroxyl group of an internal nucleotide. As with group I introns, the splicing of group II introns occurs via two transesterification steps, one involving cleavage of the 5' splice site and the second resulting in cleavage of the 3' splice site and ligation of the two exons. For example, 5' splice site cleavage results from nucleophilic attack by the 2'-hydroxyl of an internal nucleotide (typically an adenosine) located upstream of the 3' splice site, causing the release of the 5' exon and the formation of a lariat intermediate (so called because of the branch structure of the 2',5' phosphodiester bond thus produced). In the second step, the 3'-end hydroxyl of the upstream exon makes a nucleophilic attack on the 3' splice site. This displaces the intron and joins the two exons together.
Eukaryotic nuclear pre-mRNA introns and group II introns splice by the same mechanism; the intron is excised as a lariat structure, and the two flanking exons are joined. Moreover, the chemistry of the two processes is similar. In both, a 2 hydroxyl group within the intron serves as the nucleophile to promote cleavage at the 5' splice site, and the 3' hydroxyl group of the upstream exon is the nucleophile that cleaves the 3' splice site by forming the exon-exon bond. However, in contrast to the conserved structural elements that reside within group I and II introns, the only conserved features of nuclear pre-mRNA introns are restricted to short regions at or near the splice junctions. In yeast, these motifs are (i) a conserved hexanucleotide at the 5' splice, (ii) an invariant heptanucleotide, the UACUAAC Box, surrounding the branch point A, (iii) a generally conserved enrichment for pyrimidine residues adjacent to the invariant AG dinucleotide at the 3' splice site. Further characteristics of nuclear pre-mRNA splicing in vitro that distinguish it from autocatalytic splicing are the dependence on added cell-free extracts, and the requirement for adenosine triphosphate (ATP). Another key difference is that nuclear pre-mRNA splicing generally requires multiple small nuclear ribonucleoproteins (snRNPs) and other accessory proteins, which can make-up a larger multi-subunit complex (splicesome) that facilitates splicing.